Gene Tech Flashcards
Genome definition
entire set of dna including all the genes in an organism
How does gene sequencing work
only works on dna fragments so need to chop up genome first, sequence smaller pieces then build it up togethe
Proteome definition
all of the proteins that are made by the organism
Difference between sequencing genomes in simple organisms vs complex organisms
simple organisms dont have much non coding dna therefore easy to determine proteome from dna genome, but complex have large sections of non coding dna and regulatory genes therefore more difficult to sequence proteome.
rDNA tech definition
involves transferring a dna fragment from one organism to another
What are the three methods for making dna fragments
- reverse transcriptase
- using restriction enzymes
- using a gene machine
How does using reverse transcriptase work to make dna fragments?
Cells that produce the protein coded for by the target gene will contain many mRNA molecules comp. to the gene.
mRNA is isolated from cells, mixed with free nucleotides and reverse transcriptase, which uses mRNA as template to synthesise cDNA strands.
How does using restriction enzymes work to make dna fragments?
Restriction enzymes recognise palindromic recognition sequences and cut them with sticky ends.
Different res. enzymes cut different recognition sequences because the recognition sequences shape is comp. to the enzymes AS
Both the DNA and vector cut with the same enzyme with sticky ends that are complementary.
How does using the gene machine work to make dna fragments?
Sequence required is designed by computer
First nucleotide in sequence is fixed to support bead
Nucleotides are added step by step in correct order to build up short DNA sections called oligonucleotides
Oligonucleotides are joined together to form longer fragments
What are the two methods of amplifying fragments?
In vivo - using living organism
In Vitro - made outside living organisms using PCR
What are the 3 steps of in vivo cloning
Making rDNA
Transforming Cells
Identifying Transformed Cells
How to make rDNA in in vivo cloning
Locate vector DNA (plasmid) and cut using same restriction enzyme that was used to isolate DNA fragment, so sticky ends will both be complementary
Vector and DNA fragment mixed with DNA ligase to join sticky ends together = rDNA
How to transform cells in in vivo cloning
Place cells in ice cold DNA to permeate cell walls, add plasmids and give mixture heat shock
How to identify transformed cells in in vivo cloning
Insert marker genes into vectors at same time as gene is cloned
Transformed cells will grow and divide in agar where all colonies contain marker gene
eg/ Antibiotic resistance, so transformed cells wont die in antibiotic agar
eg/ Fluorescent marker gene, so only transformed cells will fluoresce in UV light
How do you ensure transformed host cells produce the protein codes for by DNA fragment
make sure vector contains specific promotor and terminator regions to tell RNA polymerase to stop and start producing mRNA
